Fibroblast activation protein regulates natural killer cell migration, extravasation and tumor infiltration

Natural killer (NK) cells play a critical role in physiologic and pathologic conditions such as pregnancy, infection, autoimmune disease and cancer. In cancer, numerous strategies have been designed to exploit the cytolytic properties of NK cells, with variable success. A major hurdle to NK-cell focused therapies is NK cell recruitment and infiltration into tumors. While the chemotaxis pathways regulating NK recruitment to different tissues are well delineated, the mechanisms human NK cells employ to physically migrate are ill-defined. We show for the first time that human NK cells express fibroblast activation protein (FAP), a cell surface protease previously thought to be primarily expressed by activated fibroblasts. FAP degrades the extracellular matrix to facilitate cell migration and tissue remodeling. We used novel in vivo zebrafish and in vitro 3D culture models to demonstrate that FAP knock out and pharmacologic inhibition restrict NK cell migration, extravasation, and invasion through tissue matrix. Notably, forced overexpression of FAP promotes NK cell invasion through matrix in both transwell and tumor spheroid assays, ultimately increasing tumor cell lysis. Additionally, FAP overexpression enhances NK cells invasion into a human tumor in immunodeficient mice. These findings demonstrate the necessity of FAP in NK cell migration and present a new approach to modulate NK cell trafficking and enhance cell-based therapy in solid tumors.


Introduction
Natural killer (NK) cells are innate lymphoid cells that in uence many physiologic and pathologic conditions through their effector and regulatory functions 1 .NK cells are canonically known to recognize and kill aberrant cells, such as virus-infected or malignant cells, using a complex detection system comprised of multiple inhibitory and activating receptors 1 .Beyond their roles as effector cells, NK cells regulate the functions of other cell types, including dendritic cells, T cells, B cells and endothelial cells, through the release of immunomodulating cytokines [2][3][4][5][6] .Due to their central role in the immune system and disease etiologies, efforts to manipulate NK cell activity have long been sought and developed to improve patient outcomes across many medical elds.
In cancer, patients with high tumoral NK cell content and activation have improved survival 7,8 and response to immunotherapy [9][10][11] .Thus, NK cells are emerging as major targets to promote cancer immunotherapy 12 .Current NK-focused immunotherapy approaches include autologous or allogeneic NK cell transfer 13 , chimeric antigen receptor-engineered (CAR) NK cells 14 , NK cell immune checkpoint inhibitors 15 , bi-or tri-speci c killer engagers (BiKEs and TriKES) 16 , and cytokine super-agonists 17 .An impediment to these therapies is inadequate NK cell homing to and/or in ltration into solid tumors.
Strategies that increase NK cell in ltration into tumors represent plausible ways to enhance NK cellrelated antitumor immunotherapies.Such work has focused almost entirely on modulating NK chemokine receptors and chemoattractants 18,19 .However, lymphocyte migration depends on more than just chemotaxis.For NK cells to successfully in ltrate any tissue, including solid tumors, they must traverse complex microenvironments (e.g., extravasation from blood vessels and navigation through dense extracellular matrices) 20 .Beyond the chemokine/chemoattractant system, little is known about the mechanisms NK cells employ to physically migrate through these tissues.
Here we describe for the rst time that human NK cells express broblast activation protein (FAP).FAP is a transmembrane serine protease primarily expressed on activated broblasts during wound healing or pathological conditions such as brosis, arthritis, and cancer 21 .FAP is primarily known for its extracellular matrix (ECM) remodeling capabilities due to its collagenase activity.Since FAP is overexpressed in diseased tissue, yet mostly absent from healthy tissue 21 , it is a promising therapeutic target in conditions like cardiac brosis 22 and cancer 23 .
After identifying FAP expression by human NK cells, we used computational approaches to elucidate the function of FAP in NK cells and validated these computational ndings in vitro using 2D assays.We then explored the impact of genetic manipulation and pharmacologic inhibition of FAP on NK cell migratory properties, extravasation, and tumor in ltration.We found that pharmacologic inhibition or deletion of FAP restrict NK cell migration, extravasation, and invasion through matrix.Conversely, forced overexpression of FAP signi cantly promotes NK cell invasion through matrix in both transwell and tumor spheroid assays, ultimately enhancing tumor cell lysis.Additionally, FAP-overexpressing cells showed a signi cantly enhanced ability to in ltrate tumors in vivo.These ndings demonstrate the necessity of proteolytic migration in NK cell function and provide an entirely new way to enhance the anti-tumor activity of NK cells.The elucidation of FAP's role in enhancing NK cell migration and tumor in ltration presents a promising avenue for the development of novel immunotherapeutic strategies in cancer treatment, potentially improving the e cacy of NK-cell based therapies.

Results
Human natural killer cells express broblast activation protein (FAP) NK cells were not previously known to produce FAP; however, we detected FAP expression at the protein level in NK92 cells and three additional human NK cell lines: NKL, YT and KHYG-1 (Fig. 1A and Fig. S1 C  and D).To exclude the possibility that FAP expression was speci c to NK cell malignancies, we assessed FAP expression in NK cells isolated from PBMCs of ve different healthy human donors and found robust FAP expression in all donor NK cells (Fig. 1B and Fig. S1E).To determine if additional human immune cell types express FAP, we assessed multiple different human B, T and monocyte cell lines for FAP expression by Western blot and found heterogeneous protein expression (Fig. 1C).This cell-line speci c FAP protein expression was consistent with FAP mRNA expression as determined by analysis of RNAseq data derived from the cancer cell line encyclopedia 24 (Fig. S1F).While we saw heterogeneous expression of FAP in B, T and monocyte cell lines, we did not detect FAP expression in healthy donor PBMC-derived B cells (CD19 + ), T cells (CD3 + ), and macrophages (CD14 + ) (Fig. 1D and Fig. S1G).Thus, FAP expression in non-NK cell lines is likely driven by their malignant biology, since FAP can be upregulated during the process of malignant transformation 21 .To further support our Western blot data, we con rmed FAP protein expression was detected on NK92 as well as normal healthy donor NK cells by immuno uorescence (Fig. 1F).
Canonically, FAP is surface-expressed, so we examined FAP expression on the NK cell surface.In order to assess this, we biotinylated cell surface proteins, and then excluded them from the cell lysate via magnetic separation.We then determined that FAP is present in total cell lysate but absent from the intracellular protein lysate (Fig. 1E), demonstrating that FAP is indeed expressed on the NK cell surface.

In NK cells, FAP gene expression correlates with extracellular matrix and migration regulating genes
We leveraged transcriptional analysis to further determine FAP's function in human natural killer cells.In 2011, Iqbal et al. performed a gene expression array on multiple NK cell lymphoma samples and NK cell lines 25 .Using these data, we assessed FAP expression in 22 NK cell lymphomas and 11 NK cell lines (Fig. 2A) and performed a correlation analysis to assess the genes that were most positively and negatively correlated with FAP expression (Fig. 2B).The top 19 genes that were most positively correlated with FAP expression are shown in Fig. 2C.We then performed GO enrichment analysis of these genes and determined that the pathways most positively correlated with FAP expression were related to ECM remodeling and cellular migration (Fig. 2D).This is consistent with the current understanding of FAP function, which is to cleave ECM components such as collagen and enhance cellular migration/invasion 21 .It is also interesting that matrix metalloproteases (MMPs) were among the top 19 genes positively correlated with FAP expression.MMPs regulate rat, mouse and human NK cell migration into collagen or Matrigel in vitro [26][27][28] .These data suggest that FAP may also regulate NK cell migration.

Manipulation of FAP regulates human NK cell migration on matrix
Based on the computational analysis, we hypothesized that FAP was expressed by human NK cells to enhance migration.To test this hypothesis, we initially compared primary NK cell migration ex vivo in the presence and absence of an FAP-speci c inhibitor (Cpd60) 29 that inhibited FAP but not FAP's most closely related protein, DPPIV (Fig. 3A) or the other members of the prolyl oligopeptidase family S9 29 .Cpd60 had no effect on NK cell viability (Fig. S2A).We then cocultured primary NK cells with EL08.1D2 cells, which have previously been shown to support spontaneous NK cell migration 30,31 and produce ECM 32 , and live imaged them for 24 h capturing photos every 2 minutes (Fig. 3B).From these time-lapse videos we manually tracked NK cell migratory paths (Movie S1 and S2).These experiments were repeated with NK cells from three different donors, with similar results.We found that FAP inhibition with Cpd60 signi cantly reduced NK cell velocity (Fig. 3E) and the accumulated distance traveled by NK cells (Fig. 3F).
These ndings were con rmed using an FAP knockout NK92 cell line.FAP was knocked out (FAP KO) in NK92 cells via a CRISPR-Cas9 system.This knockout was con rmed by Western blot and rt-qPCR (Fig. S3A and B).Similar to primary cells, NK92 cells were incubated on a con uent layer of EL08.1D2 stromal cells and imaged at 2 min intervals for 1-3 hours.Instead of manual tracking, cells were segmented and tracked using automated detection and tracking as described in Methods.Because of this, we were able to get data from 800-1800 cells per condition.We found that FAP KO cells displayed signi cantly longer arrest coe cients, de ned as the frequency of time cells were found in arrest, (Fig. 3E), slower speed (Fig. 3F), and lower accumulated distance (Fig. 3G).
We hypothesized that if FAP KO reduced NK cell migration then FAP overexpression would increase migration.We generated a FAP-overexpressing NK92 cells line (FAP OE) using retroviral transfection.These cells were selected via the GFP expression conferred by the plasmid (Fig. S4A).This upregulation was con rmed by Western blot and RT-qPCR (Fig. S4B and C).As hypothesized, FAP OE NK92 cells displayed signi cantly shorter arrest coe cients (Fig. 3E), faster speed (Fig. 3F), and longer accumulated distance (Fig. 3G).
FAP manipulation regulates the invasion of human NK cells through matrix.
We then examined the impact of FAP on NK cell invasion through matrix.To analyze this, NK92 cells (NK92, FAP KO, FAP OE) were plated in the top well of a transwell chamber.CXCL9, a known NK cell chemoattractant 33 , was placed in the lower well to stimulate NK cell invasion.NK cells were allowed to invade through the membrane coated with a Matrigel barrier for 24 hours (Fig. 4A).Notably, FAP OE in NK92 cells resulted in an almost three-fold increase in invasion through Matrigel.(Fig. 4C).Additionally, knockout as well as inhibition of FAP enzymatic activity with Cpd60, an FAP speci c inhibitor, resulted in a signi cant decrease in invasion through the Matrigel (Fig. 4B).No additional decrease was seen following treatment of FAP KO cells with Cpd60, suggesting that the decrease seen in response to Cpd60 is due to the inhibition of FAP (Fig. S5).
To verify these ndings, we performed a droplet assay.2,000 NK cells (NK92, FAP KO, and FAP OE) were plated on one side of a four well LabTek plate with CXCL9 plated on the other side of the well.They were then covered in ECM and allowed to invade for 24 hours (Fig. 4D).FAP KO in NK92 cells signi cantly reduced invasion through matrix while FAP OE in NK92 cells signi cantly increased invasion through matrix (Fig. 4E).

FAP inhibition reduces NK cell extravasation in vivo
We next set out to determine if FAP altered NK cell migratory behaviors in vivo.Since we could not detect FAP expression in murine NK cells (Fig. S1H) we opted to use zebra sh-a novel in vivo model that allows us to monitor human NK cell migratory behaviors in real-time.We injected NK92-GFP cells into the precardiac sinus of Tg(kdrl:mCherry-CAAX)y171 zebra sh embryos that express endothelial membranetargeted mCherry (Fig. 5A).Immediately after injection, NK cells migrated via the circulation to the caudal hematopoietic tissue (Fig. 5B) gradually disseminating throughout the rest of the zebra sh vasculature.
Using confocal live-imaging, with images taken approximately every 3 minutes, we captured an NK cell crawling along the inside of the blood vessel, searching for an appropriately sized pore just prior to extravasation (Fig. 5C and Movie S3, Fig. S6).After con rming that human NK cells could migrate throughout and extravasate from zebra sh vasculature, we tested the effects of FAP inhibition on NK cell extravasation.Since uorescent microscopy is amenable to imaging multiple sh simultaneously, we used uorescent microscopy to quantify the effects of the FAP inhibitor Cpd60 on NK cell extravasation.
We con rmed that the uorescent microscope was capable of detecting NK cell extravasation (Fig. 5D), and then imaged 20 sh injected with NK92-GFP cells, 10 of which were bathed in 10 µM of Cpd60, and 10 sh that were bathed in vehicle.We found that FAP inhibition signi cantly reduced NK cell extravasation from the blood vessels (Fig. 5E and F).
FAP manipulation regulates NK cell in ltration and lysis of PANC-1 cell clusters embedded in matrix NK cells regulate tumor growth and viability, yet relatively little is known about the mechanisms NK cells employ to invade through dense tumor-related extracellular matrices.To determine if FAP activity affects NK cell in ltration into tumors, we assessed the effect of FAP inhibition on NK cell in ltration into PANC-1 clusters embedded in matrix.To accomplish this, we plated 1,000 PANC-1 cells in low-adhesion U-bottom plates and allowed them to form clusters for 24 hours.We then embedded the clusters in matrix that consisted of 80% collagen/20% Matrigel and NK92-GFP cells, and added either 10 µM Cpd60 or vehicle to the media.We live imaged the cocultures for 24 hours, capturing images every 30 minutes.Then we xed the slides and stained for GFP by immuno uorescence to quantify the amount of NK cell in ltration into the clusters (Fig. 6A).FAP inhibition had no effect on cluster size (Fig. S7A).FAP inhibition signi cantly reduced NK92-GFP cell in ltration into PANC-1 clusters embedded in matrix (Fig. 6B, Movies S4-7).These experiments were repeated using PSCs with similar results (Fig. S8).
To determine if the reduced NK cell in ltration was accompanied by reduced tumor cell lysis, we repeated the PANC-1 and NK92 coculture experiment and stained the cells for actin using phalloidin and cleaved caspase-3 to identify apoptotic cells.Using the phalloidin stain we outlined the PANC-1 cell cluster, and then transposed the outline onto the cleaved caspase-3 images and quanti ed the intensity of cleaved caspase-3 within PANC-1 cell clusters (Fig. 6D and F).We found that FAP inhibition signi cantly reduced the amount of PANC-1 cell apoptosis (Fig. 6E) in 3D cultures, despite having no effect on PANC-1 cell apoptosis in 2D cell cocultures (Fig. S7B).To determine if FAP inhibition also reduced donor NK cell migration and tumor lysis, we repeated these experiments with NK cells from two donors.Since the range of PANC-1 cluster areas in the donor NK cell experiment was much wider than the range in the NK92 experiment (10-208 versus 12-70) we normalized the intensities in the donor NK cell experiment to the area of the cluster.In agreement with the NK92 cell experiments, FAP inhibition reduced donor NK cell lysis of PANC-1 cells in 3D (Fig. 6D) but not 2D (Fig. S7B).This demonstrates that FAP inhibition does not alter target cell lysis through direct impacts on NK cell cytotoxicity but rather via modulation of NK cell migration through matrix.
To determine whether FAP overexpression enhances NK cell invasion into these tumor spheroids, we repeated these experiments with NK92 cells and FAP OE NK92 cells.FAP OE NK92 cells showed signi cantly increased invasion into tumor spheroids and signi cantly increased cleaved caspase-3 expression (Fig. 6G).No increase in cytotoxicity in FAP OE NK92 cells was seen in 2D systems, suggesting the increase in apoptosis is due to an increase in NK cell invasion (Fig. S9).This suggests that FAP overexpression could be a method to enhance tumor in ltration by NK cells.

FAP overexpression enhances NK cell in ltration into tumors in vivo
As a proof of concept experiment, we set out to determine if NK cells engineered to overexpress FAP displayed enhanced in ltration into in a human tumor murine model.To test this, we injected NK92, FAP KO NK92, and FAP OE NK92 cells intravenously into mice bearing PANC-1 pancreatic tumors.Speci cally, we injected 1x10 6 PANC-1 cells subcutaneously into NSG mice then waited until tumors were at least 100 mm 3 in size before injecting 1x10 7 NK92, FAP OE NK92, or FAP KO NK92 cells into the tail vein (Fig. 7A).The mice were euthanized after 24 hours and tumors were collected, xed and stained with an anti-CD56 antibody.Tumors from mice injected with FAP OE NK92 cells had more than three times as many NK cells when compared to tumors from mice injected with NK92 cells (Fig. 7B,C).
Interestingly, there was no difference in invasion between the NK92 and FAP KO NK92 cells.Potentially because an effect size is too small to detect with our limited sample size.Alternatively, other known drivers of NK cell migration, such as MMPs, could potentially compensate for the loss of FAP 26,27,28 .
To assess NK cell tra cking to non-tumor locations, we collected and stained spleen, liver, and lung samples from each of the treated mice.There was no signi cant difference in NK content in any of the examined organs (Fig. S10).This suggests that under these experimental conditions NK92 cells preferentially invade into tumors rather than other organs; further supporting the notion that this technology could be implemented therapeutically.

Discussion
Here we show that human NK cells express FAP, which is a key regulator of NK cell migration, invasion, extravasation and tumor in ltration.This novel nding signi cantly broadens the existing understanding of NK cell migration and tissue in ltration, and illustrates a mechanism for NK cell extravasation from blood vessels.Our ndings reveal that both knockout and inhibition of FAP restrict NK cell tumor in ltration, and attenuate NK cell-mediated tumor cell lysis, underscoring the critical role of FAP-mediated migratory mechanisms in the anti-cancer activity of NK cells.Importantly, FAP overexpression enhances NK cell invasion through matrix, promoting tumor in ltration both in vitro and in vivo.Therefore, this work reveals novel insights into FAP biology and NK cell biology, with important implications for emerging NK cell-focused therapeutic strategies.
For extravasation or tissue invasion, cells must penetrate the basement membrane and interstitial tissue.During this process they are confronted by a 3D ECM that provides a substrate for adhesion and traction, as well as biomechanical resistance.For cells to tra c effectively through the ECM, which can offer narrow or non-existent pores for passage, leukocytes must adopt contracted shapes.Excessive cellular deformation can result in nuclear rupture that causes genomic damage, long-term genomic alterations and limited cellular survival.To circumvent nuclear damage, some cells employ proteolytic digestion to widen pores in the ECM 20 .Although proteolytic migration is considered less common in leukocytes versus other cell types, it has been documented.Zebra sh neutrophils and macrophages use proteolytic digestion for basement membrane transmigration 34 .Human neutrophils secrete elastase, a serine protease, to facilitate their endothelial transmigration 35 .
Unlike other immune cell types, there are few studies investigating the physical mechanisms driving NK cell migration.Decades-old research demonstrated that mouse and rat NK cell migration through Matrigel was dependent on MMPs 27,36,37 .More recent studies have used physiologic models.Putz et al. showed that heparinase regulated mouse NK cell in ltration into murine tumors 38 .Prakash et al. showed that granzyme B released from murine cytotoxic lymphocytes, including NK cells, enhanced lymphocyte extravasation via ECM remodeling, although it did not affect interstitial migration.They con rmed that a granzyme B inhibitor reduced human donor T cell transmigration through a Matrigel coated semipermeable membrane (i.e., Boyden chamber assay) 39 .Although these authors did not assess changes in human donor NK cell migration in response to a granzyme B inhibitor, it is reasonable to assume it would be similar to that of T cell migration, since both cell types express and release granzyme B. However, our nding that FAP is expressed in human NK cells, but not in all murine NK cells or other human immune cell types (Fig. 1), suggests that some migratory mechanisms can be cell-type and species-speci c.Unlike these previous studies that investigated either extravasation or tumor in ltration, we investigated both and found that NK cells use the same proteolytic migration strategy for basement membrane degradation/extravasation as well as tumor tissue in ltration.We further demonstrate that defects in proteolytic migration directly impair the ability of NK cells to in ltrate and lyse tumor cells.
FAP is a well-studied protein.Although once thought to be restricted to activated broblasts, FAP expression has been found in additional cell types such as epithelial tumor cells [40][41][42] , melanocytes 43 and macrophages 44,45 .In non-immune cells, FAP enhances cellular invasion 43,[46][47][48][49] .The role of FAP in macrophages is less clear.Arnold et al. showed that in murine tumors there is a FAP + minor subpopulation of immunosuppressive F4/80 hi /CCR2 + /CD206 + M2 macrophages.While this study highlighted how FAP + macrophages affect tumor growth, FAP's function in these macrophages was not described 44 .Tchou et al. identi ed FAP + CD45 + cells in human breast tumors by immuno uorescence.
They then used ow cytometry to demonstrate that a portion of these FAP + CD45 + cells were CD11b + CD14 + MHC − II + tumor associated macrophages.Since the ow cytometry panel used to categorize these FAP + CD45 + cells consisted of only macrophage markers, those data do not exclude the possibility that some of the FAP + CD45 + tumor cells were NK cells.In contrast to that study, we did not identify FAP expression in human macrophages (CD14 + cells) (Fig. 1F).However, we examined circulating cells, as opposed to cells in the tumor microenvironment.Future studies are needed to further categorize FAP expression in tumor immune cell populations, potentially using multicolor immuno uorescent staining.Additionally, more studies are needed to determine if the function of FAP in FAP + tumor macrophages is the same as we have described here in NK cells.
The nding that human NK cells express FAP (Fig. 1D) has several clinical implications for existing FAPtargeted therapies.For example, an anti-FAP/IL-2 fusion protein has been utilized in clinical trials though the results are not yet published (NCT02627274).The proposed mechanism of action of this drug is that it targets IL-2 to FAP expressing tumor stroma, thereby limiting on-target, off-site toxicities associated with IL-2 cytokine therapy.Our ndings that FAP is expressed on the NK cell surface suggests that anti-FAP/IL-2 fusion protein may also target IL-2 directly to NK cells, enhancing NK cell activation and potentially tumor clearance.
Anti-FAP CAR therapies are also in development to treat conditions such as cardiac brosis 50,22 , malignant pleural mesothelioma 51 , lung adenocarcinoma 52 and other cancers 53,54 .Our data suggest that anti-FAP CAR cells may also be useful in NK cell malignancies such as aggressive NK-cell leukemia.There are potential caveats to the clinical use of anti-FAP CAR T cells.It is plausible that an anti-FAP CAR T cell could induce NK cell lysis, resulting in NK cell leukopenia in humans, this toxicity might be missed in preclinical murine models.For cancer immunotherapy, an ideal anti-FAP CAR would be engineered to target FAP expression by broblasts while sparing NK cells.It should be noted that Gulati et al. performed the rst-in-human trial of an anti-FAP CAR T cell therapy and demonstrated that a FAP CAR T cell therapy induced stable disease for 1 year in a patient with malignant pleural mesothelioma without any treatment-terminating toxicities 51 .
Our nding that FAP regulates NK cell tissue in ltration (Figs. 6 and7) has clinical implications as well.These results imply the potential value of NK cells engineered to overexpress FAP in enhancing tumor in ltration and cell lysis.
Existing strategies aimed at enhancing NK cell in ltration into tumors rely on manipulating chemokine/receptor pathways.For example, Wennerberg et al. demonstrated that ex vivo expanded NK cells express higher levels of chemokine receptor CXCR3 than unexpanded NK cells which then demonstrated increased migration towards CXCL10 expressing melanomas 18 .Another approach that has been utilized is engineering NK cells to overexpress CXCR2, a chemokine receptor.This study showed that CXCR2 overexpressing NK cells had enhanced tra cking towards and lysis of renal cell carcinoma cells in vitro 19 .These ndings suggest that these strategies to enhance NK cell migration are feasible, however, chemokine pathway-altering strategies require not only elevated expression of the chemokine receptor on NK cells, but also secretion and maintenance of chemoattractants by the tumor.Additionally, many chemoattractants recruit multiple immune cell types, including immunosuppressive cells.For example, CXCL10 is a chemoattractant for cytotoxic T lymphocytes and NK cells, but also for regulatory T cells 56 .We postulate that the ideal migration-altering therapeutic approach would increase cytotoxic immune cell in ltration in tumor masses, without in uencing or even reducing immunosuppressive immune cell content in the TME.Since overexpressing FAP enhances NK92 cell tumor in ltration and lysis in vitro and in vivo (Figs. 6 and7), we speculate that engineering NK cells to overexpress FAP, either in autologous NK cell or NK CAR-NK therapies, could increase NK cell tumor in ltration and lysis.This approach is independent of tumor-associated factors, such as chemoattractant secretion, and would not be expected to induce the in ltration or expansion of immunosuppressive cell populations into the tumor microenvironment.Since proteolytic migration is required for NK cell killing of malignant cells (Fig. 6), the ability to alter protease expression or activity to enhance NK cell tumor in ltration represents a potentially promising approach to altering NK cell anti-tumor activity.Future studies are needed to explore the bene t of FAP-overexpressing NK cells in preclinical models and in clinical studies, and to determine what, if any, toxicities they induce.
In this study we have demonstrated that human NK cells express FAP and that FAP directly affects NK cell migration, extravasation and tumor in ltration.These ndings further the understanding of both FAP and NK cell biology.Importantly, FAP overexpression promotes the in ltration of NK92 cells into human tumor xenografts, suggesting a role for manipulating FAP expression to promote NK cell therapeutics.Future studies will determine if these novel ndings have meaningful implications for NK cell-based therapy strategies currently in development.

Generation of FAP Overexpressing Cells
Overexpression of FAP was induced in NK92 by retroviral transduction.Phoenix amphotropic cells were transfected with the pBMN plasmid containing the FAP gene (received from vectorbuilder) using Lipofectamine and Plus reagent (Life Technologies) as previously described 59 .Supernatants were collected from these cells after growing for 48 hours in Opti-MEM media (Life Technologies).The supernatant was mixed with Lipofectamine and Plus reagent and added to 2x10 6 NK92 cells in a 6-well plate.These cells were centrifuged for 45 min at 2000xg.This process was repeated two consecutive times and cells were ow sorted for GFP positivity three days after the nal transduction.

Generation of NK92 FAP Knockout Cells
Knockout of FAP in NK92 cells was accomplished by CRISPR using nucleofection, as previously described 60 .2µL of CAS9 RNP (Horizon Discovery, cat#CAS12205) and 2µL FAP sgRNA (Horizon Discovery, cat#SQ-003829-01-0002) were incubated together for 15 minutes at room temperature.The sgRNA complexes were then added to 1x10 6 NK92 cells resuspended in 16uL of P3 nucleofection buffer (Lonza).The nucleofection mixture was transferred to a 16-well strip for nucleofection in the Lonza 4D Nucleofector using the pulse code CM-138.The 20µL nucleofection mixture was then added directly to 1mL of pre-warmed NK media.This process was repeated an additional time and cells were used 72 hours later.

FAP Activity Assay
One day prior to assay, 5,000 PSCs/well were added to 96 well at clear bottom white polystyrene TCtreated microplates (Corning, cat#3610).The following day, PSC media was aspirated off and 50 µL of NK92 cells (lacking GFP) were added to each well containing PSCs at a 4:1 E:T ratio and incubated overnight at 37°C. 100 mM stock of dipeptidylpeptidase substrate (Acetyl-Aka-Gly-Pro-AFC) (Anaspec, CatAS-24126) was made by resuspending lyophilized substrate in DMSO.On the day of the assay, DMSO stock was then diluted 1:1000 in FAP activity assay buffer (50 mM Tris-BCl, 1 M NaCl, 1 mg/mL BSA, pH 7.5).A standard curve was generated using rFAP (R&D systems, 3715-SE-010).50 µL of rFAP standard ranging in concentration from 0.03125-2ug/mL was added to wells in triplicate.50 µL of substrate was added to each well and the plate was incubated for 5 minutes at 37°C.The plate was read on a PerkinElmer EnVision Multimode Plate Reader with 390-400 nm excitation and 580 − 510 nm emission wavelengths.The nal concentration of FAP per well was calculated using the standard curve.Data were compiled and assessed for statistical signi cance using GraphPad Prism 9.

PSC-NK92 Coculture Assay
PSCs were plated one day prior to assay at 100,000 cells/well in a 6 well collagen coated plate.NK92 cells were added at 1:1 or 4:1 effector to target (E:T) ratios and cocultured for 3-4 hours.Each well contained 50% v/v NK and PSC media and 1% v/v IL-2.Following incubation, nonadherent cells were collected.Adherent cells were washed 2X with PBS and then trypsinized with 0.05% trypsin.After detachment trypsin was quenched with equal volume PSC media and cells were collected, pelleted and washed 2X with PBS then resuspended in 600 µL of 1% BSA.Cells were immediately sent for nonsterile ow sorting of GFP + from GFP-using the BD FACS Aria Ilu cell sorter in the Georgetown Lombardi Comprehensive Cancer Center Flow Cytometry and Cell Sorting Shared Resource (FCSR).

Annexin V NK cell lysis study
One day prior to assay, PSCs or PANC1 cells were stained with DiI.If donor NK cells were used, they were stained with DiO prior to the experiment.Cells were then plated as described for the PSC-NK92 coculture assay.Following incubation period of 4 hours, all cells from a single well were collected and washed 2X with PBS.Samples were then processed by the FCSR using the Alexa Fluor 647 Annexin V and Sytox Blue staining (Biolegend).Flow data were analyzed using FloJo (v10.4.1) and statistics was performed using GraphPad Prism 9.

RNA Isolation and rt-qPCR
RNA was isolated using the PureLink RNA Mini Kit (Ambion, cat#12183020).The RNA concentration was measured using NanoDrop 8000 (Thermo Fisher Scienti c).cDNA was generated from 20-100 ng of RNA using the GoTaq 2-step RT-qPCR System (Promega, cat# A6110).qPCR was performed with SYBR Green on a StepOnePlus real-time PCR system (Applied Biosystems).Gene expression was normalized to HPRT and analyzed using 1/ΔCt method.
Immuno uoresence 5x10 5 cells were plated on coverslips for 2 hours in a 12-well plate.Cells were xed for 15 minutes at room temperature with 4% paraformaldehyde, washed with PBS, and permeabilized with 0.5% Triton X-100 for 15 minutes at room temperature.The cells were washed with PBS and then blocked with 1% BSA for 30 minutes.These cells were then incubated with primary antibody for 1 hour at room temperature.
Immuno uorescence was conducted using anti-FAP antibody (Santa Cruz, sc-65398) at a concentration of 1:500.The cells were washed three times with PBS.They were then incubated with Alexa Fluor 647 anti-mouse secondary antibody (Thermo sher, A21236) at a concentration of 1:1000.The cells were again washed three times with PBS.They were then incubated with DAPI diluted 1:1000 for 20 minutes at room temperature.They were again washed three times and then mounted on a slide using ProLong Antifade Mountant (Invitrogen, cat#10144).Antibody was validated with additional anti-FAP antibody (ab207178, abcam).These slides were then imaged on a Leica SP8 AOBS microscope in the LCCC Microscopy and Imaging Shared Resource (MISR).

Cell Surface Biotinylation
Cell surface biotinylation of NK92, NKL, YT and KHYG-1 cells was performed with the Pierce Cell Surface Protein Isolation kit (Thermo Scienti c, cat#89881) according to the manufacturer's protocol.In brief, 4x10 8 cells were pelleted and washed with cold PBS then incubated with EZ-LINK Sulfo-NHS-SS-biotin for 30 min at 4°C followed by the addition of a quenching solution.Another 1X10 6 cells were collected and saved for total cell western blotting.Cells were lysed with lysis buffer (500 µL) containing the complete protease inhibitor cocktail (Roche, cat#11697498001).The biotinylated surface proteins were excluded with NeutrAvidin agarose gel (Pierce, 39001).Samples were diluted 50 ug in ultrapure water supplemented with 50 mM DTT. Lysates were subjected to Western blotting with the anti-FAP antibody described above.

Gene expression analyses of NK cell lines
NK lymphoma and cell line gene expression was downloaded from GEO (GEO accession GSE19067) 25 using R version 3.6.2and read using affy in Bioconductor 61 .Non-NK cell samples were excluded from analysis.Heatmap was created using ComplexHeatMap version 2.1.1 62 .Correlation analysis was performed using limma in Bioconductor 63 .Gene set enrichment analysis was performed using GO enrichment 64 .

2D cell migration studies
2D migration studies were done as previously reported 31,32,65 .In brief, EL08.1D2 stromal cells were grown to a con uent monolayer on at-bottomed 96 well ImageLock plates (Essen Bioscience) pre-coated with 0.1% gelatin (Stemcell Technologies).For imaging primary cells, 10 µM of Cpd60 in RPMI media was added to the chamber 15 min before imaging.Freshly isolated human NK cells or 5,000 NK92 cells (NK92, FAP KO, FAP OE) were imaged in 96-well plates on the IncuCyte ZOOM Live-Cell Analysis System (Essen Bioscience) at 37°C in the phase-contrast mode (10× objective).Cells were allowed to settle for 30 min prior to beginning imaging every 2 minutes for 1-3 hours in an Incucyte Zoom using bright eld settings.

Automated cell tracking and analysis
Exported TIFF stacks from Incucyte images were segmented using the Cyto2 trained network provided by Cellpose {Stringer, 2021 #3} using a classi cation object diameter of 7. btrack {Ulicna, 2021 #11} was used to track segmented cells between frames.Data was analysed using cellPLATO 66 .A HDF5 le containing segmented masks and tracks for each cell was generated for each TIFF stack and saved.Custom Python functions were used to make 29 separate shape, migration, and clustering measurements per timepoint per cell.Cell tracks were ltered by area (40-300 µm 2 ) and by the number of timepoints a cell is segmented (5 to 1800).

Manual cell tracking and analysis
Manual tracking of live cells was done using the manual tracking feature in Fiji 67 .Tracks were plotted using the Chemotaxis plugin of Fiji.Cells that were in the eld of imaging for fewer than two frames were discarded, as were cells which were non-adherent or oating.EL08.1D2 cells were used as de facto ducial markers to ensure that neither they nor the microscope stage was drifting and causing apparent NK cell movement.Length and displacement measurements were derived directly from tracked cells and graphed using GraphPad software.Velocity data was obtained by dividing the total track length by the time of imaging.

Transwell assay
Matrigel was diluted 1:4 in NK media.50µL of this mixture was plated on the underside of a 5µm pore transwell insert (Corning, cat#CLS421).This was allowed to solidify for 20 minutes at room temperature.2x10 5 cells in 200µL media were plated in the top well of the plate.100ng/mL CXCL9 (R&D systems, cat# 392-MG) was added to 400µL media plated in the lower well of the plate.The cells were allowed to migrate for 24 hours and the number of cells in the bottom well was counted using a hemocytometer.

Droplet assay
Cells were stained with DiO prior to the experiment.2,000 cells were resuspended in 1µL of ECM mixture and plated on one end of a well on a 4 well Labtek plate (Thermo Scienti c, cat#154917).0.8ng of CXCL9 (R&D systems, cat# 392-MG) was resuspended in 2µL of ECM and plated on the other end of a well.The ECM mixture consisted of 20% growth factor reduced Matrigel (Corning, 10-12 mg/ml stock concentration, #354230) and 80% rat tail collagen type I at 3mg/mL (Gibco, A1048301).The two droplets were then covered in 75µL of ECM and was allowed to solidify for 45 minutes at 37°C.800µL of NK media was then added to the well and the cells were allowed to migrate for 24 hours.The entire slide was then imaged on the Olympus IX-71 Inverted Epi uorescent Microscope at 5X in the LCCC Microscopy Shared Resource and the number of cells that had migrated beyond the initial droplet were counted using FIJI.

Zebra sh studies
Zebra sh studies were conducted in accordance with NIH guidelines for the care and use of laboratory animals and were approved by the Georgetown University Institutional Animal Care and Use Committee.
Zebra sh husbandry, injections, and mounting was performed by the Georgetown-Lombardi Animal Shared Resource.Two day post fertilization stage Tg(kdrl:mCherry-CAAX) embryos were anesthetized with 0.016% tricaine (Sigma-Aldrich, St. Louis, MO, USA) in sh water (0.3g/L Sea Salt, Instant Ocean, Blacksburg, VA) and were injected with 100-200 NK92-GFP cells into the precardiac sinus using an air driven Picospritzer II microinjector (General Valve/Parker Hanni n) under a stereoscope.Following injection, embryos with cells in the caudal hematopoietic tissue were selected for analysis and mounted in 1.5% agarose plus 0.011% tricaine in sh water.Fish were maintained at 33°C until imaging.Confocal imaging was performed on a Leica SP8 AOBS microscope in the Georgetown-Lombardi Microscopy Shared Resource.Wide eld uorescent imaging was performed on a Keyence BZ-X inverted microscope.
Images were taken at 10X across multiple z-stacks.Z-stack images were compressed using full focus and haze reduction in Keyence BZ-X software.NK extravasation quanti cation was performed by counting the number of GFP cells outside red vasculature.NK extravasation quanti cation was performed blinded to the treatment conditions.Graphs of resulting data and statistical analysis was generated using Graphpad Prism 9.

3D cluster
3D clusters were generated, embedded and stained as previously described 68,69 .In brief, clusters were generated by plating 1,000 cells per well into 96-well Nunclon Sphera low adhesion plates (Thermo Scienti c, cat#174925) and incubated overnight at 37°C.The following day, 6 clusters were embedded into an ECM containing 2,000 NK cells and were plated into one well of a Nunc Lab-Tek II 8-well chamber slide (ThermoScienti c, cat#154534PK).The ECM mixture consisted of 20% growth factor reduced Matrigel (Corning, 10-12 mg/ml stock concentration, #354230) and 80% rat tail collagen type I at 3mg/mL (Gibco, A1048301).Cells were either imaged for the following 24 hours every 30 minutes using a Zeiss LSM800 scanning confocal microscope enclosed in a heated chamber supplemented with CO 2 or allowed to incubate overnight at 37°C.After 24 hours, cells in matrix were xed with 5.4% formalin for 1 hour, permeabilized with 0.5% Triton-X and blocked using goat serum.For invasion assays, NK-92-GFP cells were stained with anti-GFP (ThermoFisher, cat#A-11122).For the cell lysis assays, clusters were stained using anti-cleaved caspase-3 (Cell Signaling, cat#9661).Hoechst 33342, phalloidin, and secondary antibodies labeled with Alexa Fluor 488 nm, 546 nm, 647 nm, or 680 nm (Invitrogen) were used.
Animal studies 10 NSG (NOD.Cg-Prkdc Il2rg /SzJ) mice were divided into 3 groups of 3 with one kept as a negative control.We inoculated animals with 1x10 6 human PANC-1 cells by subcutaneous injection.When tumors were < 100mm, three mice were injected with 1x10 7 NK92 cells and 4,000 IU IL-2, three were inject with 1x10 7 FAP OE NK92 cells and 4,000 IU IL-2, three were inject with 1x10 7 FAP KO NK92 cells and 4,000 IU IL-2, and one was injected with saline by IV tail vein as a negative control for staining.24 hours after injection, mice were euthanized and the tumor, lung, liver, and spleen were excised.The tumors and organs were submitted to the histopathology and tissue shared resource core at Georgetown.Slides were stained using an anti CD-56 antibody (Abcam, ab133345) at 1:800.Slides were then blinded and NK cells were manually counted using Qupath.

Figure 1 Human
Figure 1

Figure 2 In
Figure 2